Fatty acid supplementation into warming solutions improves pregnancy outcomes after single vitrified‐warmed cleavage stage embryo transfers

Abstract Purpose This study aimed to examine the embryonic development of human 4‐cell stage embryos after warming with fatty acids (FAs) and to assess the pregnancy outcomes after single vitrified‐warmed cleavage stage embryo transfers (SVCTs). Methods Experimental study: A total of 217 discarded, vitrified human 4‐cell stage embryos donated for research by consenting couples were used. The embryos were warmed using the fatty acid (FA)‐supplemented solutions (FA group) or nonsupplemented solutions (control group). The developmental rate, morphokinetics, and outgrowth competence were analyzed. Clinical study: The treatment records of women undergoing SVCT in natural cycles between April and September 2022 were retrospectively analyzed (April–June 2022, control group; July–September 2022, FA group). Results Experimental study: The rate of morphologically good blastocysts was significantly higher in the FA group than in the control group (p = 0.0302). The morphokinetics during cleavage, morula, and blastocyst stages were comparable between the groups. The outgrowth was significantly increased in the FA group (p = 0.0438). Clinical study: The rates of implantation, clinical pregnancy, and ongoing pregnancy after SVCTs were significantly increased in the FA group (p = 0.0223–0.0281). Conclusions Fatty acid‐supplemented warming solutions effectively improve embryo development to the blastocyst stage and pregnancy outcomes after SVCTs.

ovarian stimulation on endometrial function; therefore, improved pregnancy outcomes are expected after FETs compared with those after fresh embryo transfers. [4][5][6] Furthermore, recent studies reported that maternal and perinatal complications could be reduced by the FETs in the natural cycle 7 but not in the hormone replacement cycle. 8 In addition, since the use of preimplantation genetic testing (PGT) has been broadly increasing, [9][10][11][12][13] the demand for FETs is expected to continue to increase in the coming years. However, a number of studies demonstrated the adverse effects of vitrification on developmental competence in oocytes and embryos. [14][15][16][17] Reducing developmental competence after vitrification is reportedly caused by the altered characteristics of cytoplasmic organelles and increased abnormalities of chromosomal segregation. [18][19][20][21] Our recent study reported that the vitrification procedure decreased the intracellular lipid content and subsequent developmental competence. 22 In addition, the supplementation of fatty acid (FA) into the warming solution increased the intracellular lipid content and improved the developmental competence by stimulating the β-oxidation pathway in mice and bovine. We also demonstrated that FA addition during warming improved the developmental competence of vitrifiedwarmed 4-cell stage embryos, leading to the increase of outgrowth competence in humans. However, in the previous study, the statistical power was weak due to the low sample number; therefore, to validate our previous findings, larger experiments are required. The previous study only performed embryo assessment by static microscopic observation; therefore, the impact of adding FA to warming solutions on embryonic morphokinetics is still unknown. Moreover, the efficacy of the FA addition in a clinical setting has not been evaluated. This study aimed to evaluate the efficiency of FA addition during warming on human embryonic development, including morphokinetics and clinical outcomes, by examining the development and morphokinetics of human 4-cell stage embryos after warming with or without FA and assessing the pregnancy outcomes after single vitrified-warmed cleavage stage embryo transfers (SVCTs). Kitazato Corporation, control group). 22 The warming procedures were carried out using the Cryotop method. Briefly, the tip of the Cryotop was dipped in a warming solution (thawing solution) at 37°C for 1 min, and the warmed embryos were transferred to a diluent solution. After 3 min, they were transferred to washing solution 1.

| MATERIAL S AND ME
After culturing for 5 min in washing solution 1, they were transferred to washing solution 2 and were cultured for 1 min. Images were captured every 10 min at 11 focal planes over 5-7 days of culture. The onset time points of the following events were recorded and analyzed: start of the 5-cell (t5), 6-cell (t6), 7-cell (t7), and 8-cell (t8) stages, initiation of compaction (tSC), completion of compaction process (tM), initiation of blastulation (tSB), and formation of full (tB) and expanded (tEB) blastocysts. 23,24 The incidence of direct cleavage, in which one blastomere cleaved into three or more blastomeres, 25 and reverse cleavage, in which two blastomeres fused into one blastomere, 26 was monitored. The amount of fragmentation during cleavage and morula stages was annotated. During the embryo peri-compaction period, excluded/extruded cells were identified only when they clearly displayed the presence of nuclei, as previously described. 27 The blastocyst quality was evaluated according to the Gardner's criteria. 28 The blastocysts graded AA, AB, BA, or BB were categorized as morphologically good blastocysts.

| Blastocyst outgrowth
To estimate the implantation capacity of blastocysts in vitro, the proportion of adhered blastocysts and the outgrowth area were examined as previously described. 29 The culture dishes were precoated with 10 μg/mL fibronectin (Sigma−Aldrich) at 4°C overnight. Next, 20 μL of NAKA ONESTEP medium was pipetted onto each drop before adding the oil overlay. After removal of the zona pellucida using acid Tyrode's solution, the blastocysts were placed individually into the drops and cultured for 96 h in a humidified incubator (Astec) at 37°C with 5% O 2 , 5% CO 2 , and 90% N 2 for the outgrowth culture assay. The embryos were designated as adhesion-initiating blastocysts when the trophoblast cells were visible and expanding outward from the blastocysts ( Figure 1).
The outgrowth area was measured at the end of culture using the NIS-Elements imaging software 2.0 (Nikon); the outer edge of the trophoblast was selected, and the outgrowth area was automatically calculated.

| Embryo transfer
Single vitrified-warmed cleavage stage embryo transfers were performed as previously described. 7 Briefly, SVCT was performed under vaginal ultrasound guidance using a specially designed soft silicone inner catheter (Kitazato Corporation); a single embryo was placed in a minimal volume in the upper part of the uterine cavity on Day 2 after ovulation in a natural cycle. Dydrogesterone (30 mg/ day; Mylan EPD G.K.) was administered orally during the early luteal phase after SVCT. Implantation was defined by the serum human chorionic gonadotropin level (>20 mIU/mL) in accordance with a previous study. 31 The clinical and ongoing pregnancy rates were defined according to the ultrasonographic observation of a gestational sac at 3 weeks after SVCT, and the observation of a fetal heartbeat was performed 5 weeks after SVCT. Early pregnancy loss and miscarriage during the first trimester were defined according to the absence of a gestational sac after implantation and the absence of a fetal heartbeat after the confirmation of a gestational sac. 32

| Statistical analysis
Statistical analyses were performed using the JMP software (SAS).
Proportions of the data were analyzed using chi-square test and  (Table 1). However, the proportion of inner cell mass with a morphological grade of C was significantly lower in the FA group than in the control group (p = 0.0069). The rate of morphologically good blastocysts per obtained blastocysts was significantly higher in the FA group than in the control group (p = 0.0302).
The rate of morphologically good blastocysts per total embryos warmed was also increased in the FA group than in the control group (p = 0.0089).

| Morphokinetics of the vitrified-warmed cleavage stage embryos
The developmental timings were comparable between the groups during the cleavage, compaction, and blastocyst stages ( Table 2).
The incidence of abnormal cleavages in the FA group was similar to that in the control group. The amount of fragmentation at 4-cell, 8-cell, and morula stage was comparable between the groups. The incidence of blastomere exclusion before compaction and blastomere extrusion after compaction was also comparable between the groups. There was no difference in the proportion of compaction patterns between the groups.

| Blastocyst outgrowth
The obtained blastocysts were used for the outgrowth assay ( Figure 3A,B). The adhesion rate to the fibronectin-coated dishes at 96 h after the outgrowth culture was comparable between the groups ( Figure 3C). However, the outgrowth area was significantly larger in the FA group than that in the control group (p = 0.0438, Figure 3D).

| Patient characteristics
The age of women and men was comparable between the control and FA groups (

| Pregnancy outcomes after SVCTs
The insemination method, number of blastomeres, and morphological grade of the transferred embryos were comparable between the control and FA groups (Table 4). However, the rates of implantation, clinical pregnancy, and ongoing pregnancy were higher in the FA group than those in the control group (p = 0.0252, p = 0.0223, and p = 0.0281, respectively). The rates of early pregnancy loss and miscarriage during the first trimester were comparable between the groups. The multivariate logistic regression analysis demonstrated that the probability of ongoing pregnancy was significantly increased in the FA group than in the control group (AOR, 1.46: 95% CI, 1.02-2.06; p = 0.0340; Table 5).
We stratified the pregnancy outcomes by the median of women's age (median, 38 years; Table S1). Although the ongoing pregnancy rates were numerically higher in the FA group than that in the control group in both young and advanced age groups, there was no difference between the groups.

| DISCUSS ION
This study demonstrated that warming human cleavage stage embryos using warming solutions supplemented with FA improved the blastocyst morphology after the embryo culture for 72 h. In addition, the morphokinetics during cleavage, morula, and blastocyst  The strength of this study was its analysis of a large dataset from a single center. In addition, the endometrial preparation method, techniques of the transfer, and culture conditions were uniform.
Therefore, potential bias owing to differences in the detailed conditions that potentially occur in multicenter data collection is unlikely.
However, our study has limitations, such as its retrospective design. In conclusion, we demonstrated that the supplementation of FA into the warming solutions improved pregnancy outcomes after SVCTs. Combined with recent technologies, including the embryo ranking based on the time-lapse data and artificial intelligence and PGT, the warming procedure should be optimized in each laboratory to maximize the clinical outcomes, shorten the treatment period, and reduce the patient burden.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest.

E TH I C S A PPROVA L
The study was an experimental and retrospective cohort study approved by the Institutional Review Board of Kato Ladies Clinic (approval number [21][22][23].

H U M A N R I G HT S S TATE M E NT S A N D I N FO R M E D CO N S E NT
All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and its later amendments. Informed consent was obtained from all patients for being included in the study.

A N I M A L S TU D I E S
This article does not contain any studies with animal subjects performed by any of the authors.